A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.     

复杂地形下风力机尾流数值模拟研究和激光雷达实验对比

以CFD数值计算和实验相结合的方法,对处于中国西南某多山地区陆上风电场的尾流特性进行研究,验证不同数值方法在复杂地形的适用性。首先采用2台激光雷达,测量目标风力机一个月内的自由来流风速和尾流廓线,在地形上坡加速效应下,不同大气稳定度下目标风力机的自由来流风速廓线均呈负梯度。然后分别采用经典致动盘和改进致动盘法,模拟目标风力机在主风向下的尾流发展。不同于只有风速与压降关系的经典致动盘法,改进致动盘法更考虑了叶片几何和气动参数(尺寸信息、攻角、桨距角、升阻力系数等)。通过与后置激光雷达尾流测试结果对比,这2种基于CFD技术的数值模拟方法,计算网格相同,计算时间相当,且均能较好地模拟因为复杂地形而引起的尾流偏转;其中改进致动盘的尾流形状与激光雷达相似,速度亏损也更接近激光雷达结果。因此,改进致动盘法更适合于复杂地形条件下风场模拟,较好平衡了计算的效率与精度。     

定子分段式永磁直线同步电机速度波动抑制方法研究

为了解决定子分段式永磁直线同步电机(PMLSM)存在的因磁阻效应、负载阻力、摩擦力、参数摄动以及动子进出定子时耦合面积变化所造成的控制性能下降的难题,根据动子和定子的耦合状态,提出一种在完全耦合阶段和动子退出定子阶段的分段式控制方法。首先,在动子与定子完全耦合阶段采用改进滑模控制器减小推力波动导致的速度波动,再通过加入扰动观测器降低滑模切换项所带来的抖振现象;在动子退出定子阶段,建立相关电磁参数与动子位置的函数关系,实时补偿由耦合面积变化引起的动子失速,使动子速度在退出时接近给定值。仿真及实验结果表明动定子完全耦合过程中的速度稳态误差为0.005 m/s,收敛时间为0.3 s,动子退出定子阶段的速度波动不超过0.04 m/s,满足定子分段式PMLSM用于长行程自动运输系统对平稳性及快速性的需求。     

Effect of paddy variety,milling status and aeration on the progeny emergence of Sitophilus oryzae L. (Coleoptera: Curculionidae)

In Sri Lanka, insects cause enormous losses in stored paddy; the rice weevil Sitophilus oryzae is a devastating insect. The magnitude of losses in stored paddy caused by insects varies with characteristics of paddy but has not yet been fully investigated. Due to the increased concerns on the use of synthetic chemicals, safer alternative pest management strategies for stored paddy are needed. Objectives of this study were to determine the effect of paddy variety, milling status and nature of aeration on the progeny emergence of S. oryzae. The experimental design was a three-factor factorial, completely randomized design (CRD).Freshly emerged S. oryzae adults were introduced to un-milled paddy or milled/polished rice belonging to traditional and improved varieties. The samples were maintained either open or airtight, and the progeny adults emerged was determined at monthly intervals. Progeny emergence was lower in the traditional varieties, un-milled paddy and under air-tight condition compared to improved varieties, milled/polished rice and aerated samples, respectively. Overall, the improved variety Bg 300 exhibited the maximum resistance to infestation by S. oryzae. Furthermore, white-colour and long-grain varieties produced lower progeny of S. oryzae than red-colour and short-grain varieties, respectively. It is concluded that the maximum protection in paddy/rice from insect infestation during storage can be achieved by using traditional varieties, stored as un-milled paddy (without dehusked) under airtight condition.     

综合改进奇异谱分解和奇异值分解的齿轮故障特征提取方法

针对齿轮故障特征微弱，在强背景噪声下难以有效提取的问题，提出了一种改进奇异谱分解（ISSD）结合奇异值分解（SVD）的齿轮故障特征提取方法。针对奇异谱分解（SSD）算法中模态参数需凭经验选取的缺陷，基于散布熵优化算法对SSD算法进行了改进，在得到既定的一组奇异谱分量的基础上，根据峭度值最大准则筛选出了最佳奇异谱分量并进行了SVD处理，采用奇异值能量标准谱自适应地确定了信号重构阶数以还原信号和提高降噪效果。最后对信号进行包络解调以提取齿轮故障特征，将所提方法运用到仿真信号和齿轮实测信号中，并同传统包络谱、SSD包络谱以及经验模态分解结合SVD(EMD-SVD)方法进行了对比分析，结果表明，所提方法的降噪和特征提取效果更佳，能够更加有效地实现齿轮故障的判别。     

透明红外硫系薄膜非均匀性检测及影响因素

薄膜非均匀性的无损检测对于制备大面积高质量的红外透明薄膜尤为重要。针对红外薄膜光学均匀性难以获取的困难,提出了一种同时获得单层透明红外薄膜厚度和折射率均匀性的无损检测方法。实验上,通过磁控溅射法在二氧化硅衬底上制备了厚度约1.4μm红外透明Ge-Sb-Se硫系薄膜,然后在该薄膜上标定出36个80μm×80μm区域,利用显微傅里叶红外光谱仪测得该36个区域的透射谱,通过分段滤波的方法滤除背景噪声,运用改进的Swanepoel方法计算得到了薄膜每一个区域的厚度和折射率,进而精确获得该薄膜的厚度和折射率均匀性,结果表明精度优于0.5%。     

基于随机模型的动车组制动模块稳健优化设计

针对目前产品优化设计中未考虑设计变量随机性和灵敏度指数对产品质量特性的影响及优化求解效率低等问题，提出一种综合考虑灵敏度指数与质量损失函数的稳健优化设计方法。通过假设设计变量的分布类型，得到设计变量的概率分布特征，将设计变量对产品质量特性的灵敏度指数与质量损失函数加权整合，并以此作为优化目标，以设计变量的容差作为约束条件，构建随机稳健优化模型。通过扩大种群数目、改进控制参数及增加惩罚因子的方法对遗传算法进行改进，结合改进的遗传算法对优化模型进行求解，得到优化模型的全局稳健最优解。以某动车组制动模块为例，采用给出的方法对其进行稳健优化设计，验证该方法的有效性与合理性。优化结果表明，该方法能够实现动车组制动模块的稳健优化设计，提高了制动模块的抗干扰能力。     

采用磁流变阻尼器的人体座椅悬架系统模糊ANFIS-PID复合控制技术研究

对座椅悬架用单出杆式磁流变阻尼器进行阻尼特性试验,并借助MATLAB多项式拟合工具箱对改进多项式模型中未知参数进行辨识。分析人体振动特性,建立五自由度人体座椅悬架模型。综合模糊控制器与自适应模糊神经推理系统(Adaptive Neuro-Fuzzy Inference System,ANFIS)整定PID控制器的优点,提出一种模糊ANFIS-PID复合控制策略。采用正弦信号作为外界激励,分别对被动悬架、传统模糊控制、ANFIS-PID控制及模糊ANFIS-PID复合控制人体座椅悬架系统进行仿真分析。结果表明,辨识得出的参数和建立的改进多项式模型均可满足后续仿真要求;采用模糊ANFIS-PID复合控制策略的隔振效果明显优于传统模糊控制及ANFIS-PID控制,能有效改善人体座椅悬架系统的行驶平顺性及驾乘人员乘坐舒适性。     

改良森吉米尔法退火炉中带钢的氧化研究

总结了带钢经过改良森吉米尔退火炉后的4种氧化状态,通过现场实际生产经验的总结和数据积累,分析了带钢产生表面氧化的关键因素,指出了实际操作中应该注意的事项,为以后的设计调试及生产提供了参考。     

基于改进ISODATA的无先验扩展目标聚类分析算法

基于弹载雷达多扩展目标检测的应用需求,在CFAR检测输出的基础上,对检测结果的聚类分析方法进行了论证分析,提出了改进的ISODATA算法。该算法摆脱了常用聚类分析算法对目标个数、聚类初值等先验信息的要求以及孤立点的影响。数字仿真实验验证了新算法的有效性。